ABSTRACT Conventional and targeted therapies have had little success in eradicating myeloid malignancies including in myeloproliferative neoplasms (MPNs). The inability to reliably prevent the generation of a small subset of drug-resistant stem cell-like leukemic cells (or leukemic stem cells (LSCs)) that cause relapse and the incapacity to target LSC has contributed to this failure. Mitochondrial metabolism has been implicated in regulating both LSC and hematopoietic stem cells (HSC) activity, however many other aspects of mitochondrial functions that contribute to the health of stem cell machinery remain largely unknown. We have discovered mitochondrial heterogeneity in a highly purified population of primitive HSC. Overall our results indicate that mitochondrial heterogeneity might subdivide stem cell compartment into fractions with distinct properties and activities. In addition, we have shown that the transcription factor FOXO3 that is required for both normal hematopoietic and leukemic stem cell maintenance is essential for HSC mitochondrial metabolism. Based on our studies and the similarities of normal blood-forming and leukemic stem cells, we propose to test the hypothesis that deregulated FOXO3 activity promotes the generation of pre-leukemic stem cells in the context of myeloid malignancies. We propose to test this hypothesis in a model of MPN that designate a group of blood clonal stem cell disorders that have the potential to progress to leukemia and in which metabolic/mitochondrial pathways have been broadly implicated. We will take advantage of mitochondrial heterogeneity to identify subpopulations of HSC and LSC with distinct stem cell properties and potential. Aim 1: To investigate functional consequences of Long-term-HSC mitochondrial heterogeneity; Aim 2: To elucidate the mechanism of FOXO3 regulation of mitochondria in stem cells. These studies are highly likely to improve our understanding of leukemic stem cell biology and the contribution of mitochondria to the LSC generation.